A major barrier to a better understanding of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) has been the heterogeneity within the condition and the lack of biomarkers to characterize disease phenotypes and analyze treatment outcome. While the etiology and pathogenesis of ME/CFS are poorly understood, there is evidence that immune system abnormalities are associated with symptoms in a substantial number of affected individuals. In addition, many ME/CFS patients complain of gastrointestinal (GI) symptoms of unknown etiology. However, immune responses to dietary and microbial antigens as underlying causes of the reported GI symptoms or as contributors to systemic inflammation have not been explored in controlled studies. Our preliminary data from a study of well-characterized patients and controls demonstrate that individuals with ME/CFS exhibit significantly elevated antibody reactivity to gluten, which correlates with the severity of GI symptoms. Moreover, the results show that the observed immune response to gluten in ME/CFS is fundamentally distinct from that in celiac disease, being independent of the action of transglutaminase 2 enzyme and HLA-DQ2/DQ8 molecules. Additional data within this application indicate that increased antibody reactivity to gluten can be associated with microbial translocation in individuals without celiac disease. We hypothesize that the molecular targets of the antibody response to gluten in ME/CFS are unique, further characterization of which may identify novel biomarkers of the condition, and that this immune response is associated with microbial translocation and systemic inflammation in a subset of ME/CFS patients. To analyze the molecular targets of the identified immune reactivity and assess its implications for ME/CFS, we propose two specific aims. In Aim 1, we will map the antigenic specificity of the immune response to gluten in ME/CFS through mass spectrometry-assisted proteome analysis and epitope mapping. In Aim 2, we will assess the relationship between the immune response to gluten and specific markers of microbial translocation and systemic inflammation in ME/CFS. The information that is expected to emerge if the aims of the proposed project are achieved would 1) offer biomarkers that may be useful in identifying subsets of patients or individuals at risk of developing ME/CFS, 2) support the examination of specific treatment strategies targeted at the identified subset of patients, and 3) yield experimental support for closer examination of the role of intestinal barrier defects and aberrant mucosal immune response in the pathophysiology of ME/CFS. This grant proposal is in line with the program announcement's (PAR-12-033) request for applications that examine the etiology, diagnosis, pathophysiology, and treatment of chronic fatigue syndrome, such as the identification of environmental, including dietary, precipitants and the development of novel and objective biological markers for the diagnosis of ME/CFS.